Immobilization Of Invertase Research Articles

Cell wall invertase immobilization within calcium alginate beads

Yeast cell wall invertase (CWI) was immobilized within calcium alginate beads. The result of entrapment was the complete immobilization of all the added CWI. Three types of biocatalysts were prepared: CWI-S, CWI-155 and CWI-157. The optimum pH values were 4.5 and 5.0 for free and immobilized invertase, respectively. The optimum temperature was 60 °C, for both free CWI and CWI-S immobilizate. 80 °C was the optimum temperature for CWI-155 whereas the optimum temperature for CWI-157 ranged from 50 °C to 80 °C. Immobilized CWI was more stable than was free CWI above optimum activity temperatures. The activation energies were 32 kJ/mol for free CWI and 45, 21 and 25 kJ/mol for CWI-S, CWI-155 and CWI-157, respectively. The K m values of free and immobilized CWI-157 were 28.4 mM and 72 mM, respectively. The V max values were estimated as 4.5 mM/min and 0.42 mM/min, respectively. Immobilized CWI-157 was tested in a batch reactor using 70% sucrose (w/v). Complete sucrose conversion was achieved after 55 h. After 40 consecutive cycles, CWI-157 retained 90% of its activity.

Covalent immobilization of invertase on chemically activated poly(2-hydroxyethyl methacrylate) microbeads

Properties of invertase immobilized on poly(2-hydroxyethyl methacrylate) microbeads activated by epichlorohydrin or cyanuric chloride were studied. After 20 repeated uses for 3 days, the activity of the immobilized enzyme was 92–93%.

Poly(phenylene oxide) films modified with allylamine plasma as a support for invertase immobilization

The paper describes a method for preparation of polymer support suitable for covalent invertase immobilization. Modification of poly(phenylene oxide) films by plasma polymerization of allylamine has been applied to introduce amine functionality on the polymer surface. It has been observed that the polymer surface became covered in plasma by a loosely fixed, moderately hydrophilic layer that should be removed before the immobilization process. The chemical character of the stable sub-layer has been related to several modification parameters: geometry of reactor, mode of plasma action and composition of gaseous mixture. Methods for determination of surface concentration of amine groups have also been presented and discussed from the immobilization point of view.

Direct immobilization of a recombinant invertase to Avicel by E. coli overexpression of a fusion protein containing the extracellular invertase from Zymomonas mobilis and the carbohydrate-binding domain CBD Cex from Cellulomonas fimi

The Zymomonas mobilis gene invB, encoding an extracellular invertase, was translationally fused to the cellulose binding domain Cex (CBD Cex) from Cellulomonas fimi and expressed in Escherichia coli. The fusion protein obtained, termed INVB-CBD, was purified and immobilized to Avicel (crystalline cellulose) in a single step. INVB-CBD showed an invertase activity of 20.8 IU/mL and was able to bind Avicel at a ratio of 5.3 mg of protein per gram of Avicel in 5 min. Both free and immobilized INVB-CBD showed the same optimal pH (5.5) than native INVB. However, an optimal temperature shift of −10 °C was observed for both free and immobilized INVB-CBD (45 °C) compared with the native INVB (55 °C). The K m value for the free and immobilized INVB-CBD was 193 and 81 mM of sucrose, respectively; whereas, the K m value for the native INVB was 208 mM of sucrose. Thus, suggesting an improvement in the substrate affinity of INVB after the addition of the CBD Cex tag. Additionally, a decrease in the V m value was found for the free and immobilized INVB-CBD compared with the native INVB.

Invertase reversibly immobilized onto polyethylenimine-grafted poly(GMA–MMA) beads for sucrose hydrolysis

The epoxy group containing poly(glycidyl methacrylate-co-methylmethacrylate) poly(GMA–MMA) beads were prepared by suspension polymerisation and the beads surface were grafted with polyethylenimine (PEI). The PEI-grafted beads were then used for invertase immobilization via adsorption. The immobilization of enzyme onto the poly(GMA–MMA)–PEI beads from aqueous solutions containing different amounts of invertase at different pH was investigated in a batch system. The maximum invertase immobilization capacity of the poly(GMA–MMA)–PEI beads was about 52 mg/g. It was shown that the relative activity of immobilized invertase was higher then that of the free enzyme over broader pH and temperature ranges. The Michaelis constant ( K m) and the maximum rate of reaction ( V max) were calculated from the Lineweaver–Burk plot. The K m and V max values of the immobilized invertase were larger than those of the free enzyme. The immobilized enzyme had a long-storage stability (only 6% activity decrease in 2 months) when the immobilized enzyme preparation was dried and stored at 4 °C while under wet condition 43% activity decrease was observed in the same period. After inactivation of enzyme, the poly(GMA–MMA)–PEI beads can be easily regenerated and reloaded with the enzyme for repeated use.

Immobilization of invertase and glucose oxidase in conducting copolymers of thiophene functionalized poly(vinyl alcohol) with pyrrole

In this study, immobilizations of invertase and glucose oxidase were achieved in conducting thiophene functionalized copolymers of vinyl alcohol with thiophene side groups and pyrrole (PVATh/PPy) via electrochemical polymerization. The kinetic parameters, V max (maximum reaction rate) and K m (substrate affinity), of both free and immobilized enzymes were determined. The effect of supporting electrolytes, p-toluene sulfonic acid and sodium dodecyl sulfate, on the enzyme activity and film morphologies was examined. The optimum temperature, operational and storage stabilities of immobilized enzymes were determined. PVATh/PPy copolymer was found to exhibit significantly enhanced properties compared to pristine polypyrrole.

Novel method of enzymes stabilization on crosslinked thermosensitive carriers

In this paper an immobilization of invertase on thermosensitive copolymers of N-isopropylacrylamide and 2-hydroxyethyl methacrylate or glycidyl methacrylate modified by aminolysis is evaluated. The method is based on the swelling properties of stimuli-sensitive polymers, which work like a pump that sucks up the enzyme on cooling and then on subsequent crosslinking of the enzyme. The attention was focused on the properties of the carrier–enzyme systems, particularly on the effect of crosslinking on their stability. Activity of TG8-NH 2 carrier was very low and independent on concentration of glutaraldehyde used, but carriers TH8 and TH8-NH 2 were more active, especially when 1.0 and 2.5 vol.% of glutaraldehyde were used. It was also observed, that preparations crosslinked by glutaraldehyde were more stable than preparations without crosslinking agent.

Immobilization of chitosan-modified invertase on alginate-coated chitin support via polyelectrolyte complex formation

Saccharomyces cerevisiae invertase was chemically modified with chitosan and further immobilized on sodium alginate-coated chitin support. The yield of immobilized protein was determined as 85% and the enzyme retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 °C and its thermostability was enhanced by about 9 °C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was four-fold more resistant to thermal treatment at 65 °C than the native counterpart. The biocatalyst prepared retained 80% of the original catalytic activity after 50 h under continuous operational regime in a packed bed reactor.

Mercury–enzyme inhibition assays with an amperometric sucrose biosensor based on a trienzymatic-clay matrix

The feasibility of amperometric sucrose and mercury biosensors based on the immobilization of invertase, glucose oxidase and mutarotase entrapped in a clay matrix (laponite) was investigated. The enzyme clay gel was deposited onto platinum electrodes and cross-linked with glutaraldehyde. The response of the biosensors to sucrose was measured by potentiostating the modified electrodes at +0.60 V versus Ag/AgCl in order to oxidize the enzymatically generated hydrogen peroxide. Mercury concentration was measured by inhibiting the invertase activity. The effect of different parameters, such as pH of the analyte solution, enzyme ratio, mercury incubation time, on the biosensor performance towards sucrose and mercury determinations was studied. The biosensor inhibition by inorganic and organic mercury was evaluated and the study of interference from other metal ions revealed a good selectivity towards mercury. Under optimized conditions, sucrose was determined from 3.3 × 10 −7 to 5.1 × 10 −3 M and mercury(II) in the concentration range of 1 × 10 −8 to 1 × 10 −6 M.

Immobilization of invertase in conducting polypyrrole/PMMA‐co‐PMTM graft copolymers

AbstractIn this study, invertase was immobilized in copolymer electrodes constructed. Three different types of polymethyl methacrylate‐co‐polymethyl thienyl methacrylate matrices were used to obtain copolymers that were characterized by FT‐IR spectroscopy. Immobilization of enzymes was carried out by the entrapment of the enzyme in conducting polymer matrices during electrochemical polymerization of pyrrole through thiophene moieties of polymers. Immobilization of the enzyme was achieved by application of 1.0 V constant potential on a platinum electrode for 30 min in solution. The effects of temperature and pH on the activity of the enzyme electrodes were examined and operational stability studies were done. The changes in the maximum reaction rate and the variations in the Michaelis–Menten constant were studied. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 502–507, 2005

Immobilization of invertase onto dimer acid‐co‐alkyl polyamine

AbstractInvertase was immobilized onto the dimer acid‐co‐alkyl polyamine after activation with 1,2‐diamine ethane and 1,3‐diamine propane. The effects of pH, temperature, substrate concentration, and storage stability on free and immobilized invertase were investigated. Kinetic parameters were calculated as 18.2 mM for Km and 6.43 × 10−5 mol dm−3 min−1 for Vmax of free enzyme and in the range of 23.8–35.3 mM for Km and 7.97–11.71 × 10−5 mol dm−3 min−1 for Vmax of immobilized enzyme. After storage at 4°C for 1 month, the enzyme activities were 21.0 and 60.0–70.0% of the initial activity for free and immobilized enzyme, respectively. The optimum pH values for free and immobilized enzymes were determined as 4.5. The optimum temperatures for free and immobilized enzymes were 45 and 50°C, respectively. After using immobilized enzyme in 3 days for 43 times, it showed 76–80% of its original activity. As a result of immobilization, thermal and storage stabilities were increased. The aim of this study was to increase the storage stability and reuse number of the immobilized enzyme and also to compare this immobilization method with others with respect to storage stability and reuse number. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 1526–1530, 2004

Immobilization of invertase and glucose oxidase in poly 2-methylbutyl-2-(3-thienyl) acetate/polypyrrole matrices

Immobilization of invertase and glucose oxidase in conducting polypyrrole and copolymers of poly 2-methylbutyl-2-(3-thienyl) acetate with pyrrole were achieved via electrochemical method. Sodium dodecyl sulphate was found to be the most suitable supporting electrolyte. Maximum reaction rate, Michaelis–Menten constant and optimum temperatures were determined for native and immobilized enzymes. Storage and operational stabilities of enzyme electrodes were also investigated.

Immobilization of invertase and glucose oxidase in conducting H-type polysiloxane/polypyrrole block copolymers

In this study, immobilizations of enzymes, invertase and glucose oxidase, were achieved in conducting copolymers of N-pyrrolyl terminated polydimethylsiloxane/polypyrrole (PDMS/PPy) matrices via electrochemical polymerization. The kinetic parameters, v max (maximum reaction rate) and K m (substrate affinity), of both free and immobilized enzymes were determined. The effect of supporting electrolytes, p-toluene sulfonic acid and sodium dodecyl sulfate, on enzyme activity and film morphologies was examined. The optimum temperatures and operational stabilities of immobilized enzymes were determined. PDMS/PPy copolymer matrix was found to exhibit significantly enhanced properties compare to pristine polypyrrole in terms of relative enzyme activities, kinetic parameters and operational stabilities.

Immobilization of invertase in copolymers of thiophene functionalized menthyl ester with pyrrole

Copolymers of thiophene functionalized menthyl monomer (MM) with pyrrole were used in immobilization of invertase. Michaelis-Menten constant and maximum reaction rates for immobilized invertase were investigated. Temperature optimization. operational stability and shelf-life of obtained enzyme electrodes were studied.

Immobilization of invertase on celite and on polyacrylamide by an absorption procedure

AbstractInvertase from Saccharomyces cerevisiae was immobilized on celite and on polyacrylamide by an absorption procedure. The properties of the immobilized invertase were characterized and compared with those of soluble invertase. The activity yield for immobilized invertase on celite and on polyacrylamide was 92% and 81% respectively. The optimum pH and temperature for both soluble and immobilized invertase activity were 4.6 and 60 °C respectively. The activity of immobilized invertase is stable in the range pH 4.0–6.5. The immobilized invertase was thermostable when incubated at temperatures ranging from 40 to 60 °C. Immobilized invertase had a high stability when stored at room temperature for 90 days and had an excellent operational stability when used 20 times repeatedly. The invertase immobilized on celite was more stable than invertase immobilized on polyacrylamide. The invertase preparations immobilised by absorption procedures exhibited marked stability towards temperature, pH changes and had high storage and operational stability, suggesting their excellent potential for use as supports. Copyright © 2003 Society of Chemical Industry

Immobilization of invertase and glucoamylase on a macroporous copolymer of etyleneglycoldimethacrylate and glycidyl methacrylate and potential applications in biotechnology

The optimal conditions for the immobilization of invertase and glucoamylasewere found via their carbohydrate moiety on a macroporous copolymer of ehtyleneglycoldimethacry late and glycidylmethacrylate. Almost all of the added enzyme was bound to the polymer by increasing the time of incubation of the oxidized enzyme with polymer. A specific activity of 5500 U/g for invertase was obtained and 1100 U/g for glucoamylase. The specific productivity for invertase in a packed bed reactor was 3.5 kg/lh and for glucoamylase 1.9 kg/lh. During continuous use in a packed bed the reactor operational half life for invertase was 290 days, while no decrease in activity was observed for glucoamylase. In 50% (v/v) ethanol the immobilized enzymes were five to ten times more stable, and more than 200 times more stable in 25% (v/v) dioxane. The immobilized enzymes retained all activity in petroleum ether after 3 days of incubation. Because of their higher stability over native enzymes, and the large surface area of the polymer immobilized glucoamylase and invertase could be more useful for glycoside synthesis in non-aqueous solvents than native ones.

Immobilization of invertase in conducting copolymers of 3-methylthienyl methacrylate

Immobilization of invertase in conducting copolymer matrices of 3-methylthienyl methacrylate with pyrrole and thiophene was achieved by constant potential electrolysis using sodium dodecyl sulfate (SDS) as the supporting electrolyte. Polythiophene (PTh) was also used in entrapment process for comparison. Kinetic parameters, Michaelis–Menten constant, K m, and the maximum reaction rate, V max, were investigated. Operational stability and temperature optimization of the enzyme electrodes were also examined.

Reversible immobilization of invertase on Sepabeads coated with polyethyleneimine: optimization of the biocatalyst's stability.

Invertase from S. cerevisiae has been immobilized by ionic adsorption on Sepabeads fully coated with PEI. The enzyme was strongly adsorbed on the support (no desorption of the invertase was found under conditions in which all of the enzyme was released from conventional anionic exchanger supports (e.g., DEAE-agarose)). Nevertheless, the enzyme could still be desorbed after its inactivation, and new fresh enzyme could be adsorbed on the supports without detrimental effects on enzyme loading. This is a multimeric enzyme, its minimal oligomerization active state being the dimer, but under certain conditions of pH and concentration it may give larger multimers. Very interestingly, results suggested that the adsorption of the enzyme on this large and flexible polymeric bed was able to freeze some of the different oligomeric structures of the enzyme. Thus, we have found that the enzyme immobilized at certain pH values (pH 8.5) and high enzyme concentration, in which the main enzyme structure is the tetramer, was more stable than immobilized preparations produced in conditions under which oligomerization was not favorable (dimers at low enzyme concentration) or it was too high (e.g., hexamers-octamers at low pH value). The optimal enzyme preparation remained fully active after a 15-day incubation at 50 degrees C and pH 4.5 (conditions of standard industrial use) and presented an optimal temperature approximately 5 degrees C higher than that of soluble enzyme.

Immobilization of invertase onto crosslinked poly(p‐chloromethylstyrene) beads

AbstractInvertase was immobilized onto poly(p‐chloromethylstyrene) (PCMS) beads that were produced by a suspension polymerization with an average size of 186 μm. The beads had a nonporous but reasonably rough surface. Because of this, a reasonably large external surface area (i.e., 14.1 m2/g) could be achieved with the proposed carrier. A two‐step functionalization protocol was followed for the covalent attachment of invertase onto the bead surface. For this purpose, a polymeric ligand that carried amine groups, polyethylenimine (PEI), was covalently attached onto the bead surface by a direct chemical reaction. Next, the free amine groups of PEI were activated by glutaraldehyde. Invertase was covalently attached onto the bead surface via the direct chemical reaction between aldehyde and amine groups. The appropriate enzyme binding conditions and the batch‐reactor performance of the immobilized enzyme system were investigated. Under optimum immobilization conditions, 19 mg of invertase was immobilized onto each gram of beads with 80% retained activity after immobilization. The effects of pH and temperature on the immobilized invertase activity were determined and compared with the free enzyme. The kinetic parameters KM and VM were determined with the Michealis–Menten model. KM of immobilized invertase was 1.75 folds higher than that of the free invertase. The immobilization caused a significant improvement in the thermal stability of invertase, especially in the range of 55–65°C. No significant internal diffusion limitation was detected in the immobilized enzyme system, probably due to the surface morphology of the selected carrier. This result was confirmed by the determination of the activation energies of both free and immobilized invertases. The activity half‐life of the immobilized invertase was approximately 5 times longer than that of the free enzyme. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 83: 1268–1279, 2002

Immobilization and stabilization of invertase on Cajanus cajan lectin support

Use of lectins as ligands for the immobilization and stabilization of glycoenzymes has immense application in enzyme research and industry. But their widespread use could be limited by the high cost of their production. In the present study preparation of a novel and inexpensive lectin support for use in the immobilization of glycoenzymes containing mannose or glucose residues in their carbohydrate moiety has been described. Cajanus cajan lectin (CCL) coupled covalently to cyanogen bromide activated Seralose 4B could readily bind enzymes such as invertase, glucoamylase and glucose oxidase. The immobilized and glutaraldehyde crosslinked preparations of invertase exhibited high resistance to inactivation upon exposure to enhanced temperature, pH, denaturants and proteolysis. Binding of invertase to CCL-Seralose was however found to be readily reversible in the presence of 1.0 M methyl α-D mannopyranoside. In a laboratory scale column reactor the CCL-Seralose bound invertase was stable for a month and retained more than 80% of its initial activity even after 60 days of storage at 4°C. CCL-Seralose bound invertase exhibited marked stability towards temperature, pH changes and denaturants suggesting its potential to be used as an excellent support for the immobilization of other glycoenzymes as well.